Listeria monocytogenes is a model facultative intracellular pathogen which has been widely used for the study of cell-mediated immunity. In preliminary studies on the growth of this bacterium in tissue culture cells, the sequence of events involved with intracellular growth and cell to cell spread have been defined. These studies have provided a cell- biological explanation for the absolute requirement for cell-mediated immunity, i.e. the bacteria never leave the host cytoplasm yet are able to spread cell to cell. The goal of the proposed research is to define, in molecular terms, bacterial determinants required for intracellular growth and cell to cell spread. The strategy is to isolate transposon Tn916 and Tn917-lac insertion mutations in L. monocytogenes which are either defective or altered in intracellular growth and/or cell to cell spread. Novel delivery systems of Tn917-lac will be exploited which will assist in the isolation and characterization of mutations. Three different selection schemes will be utilized to isolate mutants: 1) A novel technique called intracellular methicillin selection of mon-growing mutants; 2) isolation of small-plaque mutants; and 3) isolation of mutants which fail to plaque. The mutants will be characterized with respect to virulence in mice, cell biological defect, and/or nutritional defect. Genes encoding essential determinants of pathogenicity will be cloned and their nucleotide sequence will be determined. These genes will be cloned into an E. coli-L. monocytogenes shuttle plasmid and reintroduced into L. monocytogenes. Expression of essential determinants of pathogenesis will be studied inside cells by monitoring Beta-galactosidase expression of Tn917-lac insertion mutants. This research represents a model system to study the molecular biology of intracellular parasitism and should provide insight into the molecular requirement to induce cell-mediated immunity.